SOFTWARE AND INSTRUMENTATION TO IDENTIFY CANCER GENES

快速重新测序基因组区域或确定大量可能与癌症发展有关的基因的差异表达谱的能力对癌症研究人员来说是非常有价值的。 CGAP和人类基因组计划产生了大量的DNA序列信息，这些信息可以有效地分析，然后在当代“芯片”式重新测序或表达阵列中表示。 这严重限制了可以彻底检查的临床样品的数量。 我们建议构建专用阵列，其具有由计算机识别和设计的可立即重构的基因序列。 该阶段性创新奖将回答以下问题：1）阵列设计软件、数字光化学（DOC）芯片制造设备、MAGNA/HIC读出设备和分析/基因网络软件能否构建并加固用于癌症样本的常规分析，以通过基因表达谱和基于芯片的重测序区分癌症和非癌细胞及其进展？ 2)随着新的基因组数据的积累，DOC方法所实现的可定制阵列能否不断扩展和改进，以生成专用于分析不同癌细胞类型的芯片？ 3)通过计算机分析（如虚拟表达阵列计算）确定的候选cDNA序列（CGAP）和更大的基因组区域是否可以重新测序，以确定癌症患者人群中的信息性总体变异，直至SNP，从而确定新的致癌基因或肿瘤抑制基因？本项目的具体目标是：1）开发用于识别和阐明候选癌症相关基因的生物信息学工具，包括用于基因表达和重测序阵列的设计、读出和分析的软件; 2）开发和完成一个数字光学化学（DOC）阵列制造单元，能够合成至少100个，3）通过修改/复制内部开发的高光谱成像显微镜来开发和构建定制DOC读出系统;以及4）用于DNA微阵列芯片的设计、制造和数据分析以及随后对与癌症研究相关的临床样品进行测试的整个软件和硬件系统的集成。 用于突变检测、SNP发现、等位基因分型和表达分析的集成系统的测试将进展到使用已用新的激光捕获显微切割系统纯化的来自活检和细针抽吸物的存档和前瞻性收集的细胞。这将使我们的系统与当前的主要技术相结合，以产生一种在转化和临床试验癌症研究中广泛使用的工具。

The ability to rapidly re-sequence a genomic region or to determine the differential expression profile of a large number of genes that are potentially implicated in cancer development will be extremely valuable for the cancer researcher. There is a tremendous amount of DNA sequence information being generated by the CGAP and human genome programs, more than can be effectively analyzed and then represented in contemporary 'chip' style re- sequencing or expression arrays. This severely limits the number of clinical samples that can be thoroughly inspected. We propose to construct dedicated arrays which have immediately reconfigurable gene sequences identified and designed by computer. This phased innovation award will answer the following questions: 1) Can the array design software, Digital Optical Chemistry (DOC) chip manufacturing device, the MAGNA/HIC readout device, and analysis/gene network software be constructed and ruggedized for routine analysis of cancer samples to differentiate cancer non-cancer cells and their progression by gene expression profiling and chip based resequencing? 2) Can the customizable arrays made possible by the DOC approach be continuously expanded and improved as new genomic data is amassed to generate chips dedicated to analysis of different cancer cell types? 3) Can candidate cDNA sequences (CGAP) and larger genomic regions identified by computer analysis such as Virtual Expression Array calculations be re-sequenced to identify informative gross variations down to SNPs in cancer patient populations to identify new oncogenes or tumor suppressor genes? The specific aims of this program are: 1) To develop bioinformatics tools for the identification and ellucidation of candidate cancer related genes including software for the design, readout and analysis of gene expression and re-sequencing arrays; 2) to develop and complete a Digital Optical Chemistry (DOC) array fabrication unit capable of synthesis of at least 100,000 custom oligonucleotide array members on a single chip with the capability of rapidly constructing chips with different arrays every 2 hours; 3) to develop and construct a custom DOC readout system by modifying/replicating an in-house developed hyperspectral imaging microscope; and 4) the integration of the entire system of software and hardware for the design, fabrication, and data analysis of DNA microarray chips and their subsequent testing on clinical samples relevant for cancer research. The testing of the integrated system for use in mutation detection, SNP discovery, allelotyping, and expression analysis will progress to use archival and prospectively collected cells from biopsies and fine needle aspirates that have been purified with the new laser capture microdissection system. This will integrate our system with current major technologies to produce a tool that should be widely available in translational and clinical trials cancer research.